Male mice homozygous for a mutation in the estrogen receptor alpha gene (ERKO mice) are infertile, while those homozygous for a mutation in the estrogen receptor beta gene are fertile. The goals of this project are to determine the role of estrogen in male germ cell development, the causes of infertility in male ERKO mice, and the identity of estrogen-regulated genes in the testis and epididymis. In addition, progesterone is reported to bind to membrane receptors on human sperm to cause rapid activation of signal transduction pathways without causing effects on gene transcription. It has been hypothesized that a membrane-associated progesterone receptor (MAPR) regulates male reproductive processes. The goals are to determine where and when MAPR mRNA and protein are present in the male reproductive system, and to define the function of MAPR during spermatogenesis, sperm maturation and fertilization. It is unknown if male ERKO mice are infertile due to disruption of estrogen-regulated processes within spermatogenic cells, within associated somatic cells, or both. We hypothesized that estrogen receptors do not directly regulate germ cell development, but are required by somatic cells in the testis and/or epididymis. We addressed this by transplanting spermatogenic cells from the testes of ERKO+/- or ERKO-/- donor mice to those of isogenic wild-type mice that had been treated with busulphan to eliminate most endogenous spermatogenic cells. After allowing regeneration of spermatogenesis, recipients were mated with wild-type females. The ERKO donor mice were C57Bl/6J (black), the male recipients were albino C57BL/6J, and the wild-type females were albino CD-1. This allowed the use of coat color markers to identify offspring produced from transplanted germ cells (black) and from regenerating endogenous spermatogenic cells (white). Two litters of mice were sired recently by recipients that received germ cells from ERKO-/- donors, one with two black pups and two white pups, and one with 10 black pups. These results support the hypothesis that male germ cells do not directly require the estrogen receptor for development, and suggest that ERKO males are infertile because of functional deficiencies in somatic cells of the testis or epididymis. Other studies seek to identify genes in somatic cells of the testis and epididymis that are regulated by estrogen and lead to infertility in ERKO males. Attempts to use differential display RT-PCR for this purpose did not give useful results. A cDNA suppression subtractive hybridization method then was used to generate a cDNA library representing genes differentially expressed in the testis of ERKO and wild-type mice. Differentially expressed clones currently are being characterized, and a tissue inhibitor of metalloproteinase-2 (TIMP-2) is an early candidate for further study. When they become available, we hope to use mouse gene microarrays to identify genes that are directly or indirectly induced or down-regulated in the testis and epididymis of ERKO mice relative to wild-type mice. We will determine if differences in gene expression signatures in ERKO and wild-type mice identify the functional changes that lead to infertility in ERKO males. Other investigators hypothesized that a membrane-associated progesterone receptor (MAPR) regulates male reproductive processes. Peptide sequences were determined for an abundant protein present in a progesterone-binding complex purified from porcine liver membranes. Those sequences were used to design PCR primers and to generate a probe for screening a porcine vascular smooth muscle cDNA library. A cDNA was isolated with significant sequence homology (76%) to the 25-Dx cDNA. This cDNA was isolated from a rat liver library in the laboratory Dr. Douglas Bell at NIEHS and suggested to encode a dioxin-inducible protein. We used the rat 25-Dx cDNA to clone the mouse homolog and carried out Northern blot, Western blot, and immunohistochemical studies, focusing on the male mouse reproductive system because of the reported role of progesterone in the acrosome reaction. Because the putative MAPR lacks the features that should be part of a progesterone receptor (steroid-binding domain, calcium-binding domain, or kinase domain), we postulated that other proteins in the progesterone-binding protein complex would have these features. Yeast two-hybrid screens of mouse liver and testis libraries were carried out to identify other proteins that associate with the putative MAPR. Several different bait constructs were used, an extensive series of screens performed, and over one hundred clones sequenced. However, multiple copies of overlapping clones were not identified, as usually occurs for proteins that interact specifically, and the clones isolated did not have the features that would be predicted to be present in a progesterone-binding protein complex. The MAPR project has been shelved for the present time because of the lack of evidence of an association between the protein encoded by 25-Dx and other proteins likely to constitute a progesterone receptor.